Nervous necrosis virus (NNV) is a major worldwide viral pathogen of several economically important fish species and can cause serious damage to the aquaculture industry. NNV infection can result in rapid and total death of fish fry in commercial hatcheries, and cause disease and high morality in hatchery-reared larvae and juveniles. Typical symptoms of infection include spiral swimming behavior just before death, and infection can be confirmed histopathologically by observing vacuoles in the retina and brain tissue.
NNV is classified as member of the Nodaviridae (see e.g., Mori et al., Virology 187:368-371, 1992). The NNV genome is composed of two (+) strand RNA molecules. RNA 1 is about 1.5 kb in length and encodes a structural coat protein, and RNA 2 is about 3 kb in length and encodes an RNA polymerase.
The invention is based on the discovery of a new coat encoded by the RNA 1 segment of an NNV isolate, and the discovery that this new NNV protein is useful as a capture antigen in an NNV detection assay and as an antigen in an NNV vaccine. The amino acid sequence of this NNV protein is shown below.
In the natural NNV isolate, this amino acid sequence is encoded by the nucleotide sequence shown below.
Accordingly, the invention features a substantially pure polypeptide including an amino acid sequence at least 99% (e.g., 100%) identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the polypeptide can include the amino acid sequence of SEQ ID NO:2, with up to 4 conservative amino acid substitutions, which would not be expected to affect the ability of the polypeptide to serve as a capture antigen in an NNV detection assay or as an antigen in an NNV vaccine (e.g., a subunit or DNA vaccine).
The invention further includes an isolated nucleic acid encoding a polypeptide of the invention, a vector including a nucleic acid of the invention, or a cultured host cell containing a nucleic acid of the invention.
In other aspects, the invention includes (1) a method of producing a polypeptide by culturing a cultured host cell of the invention in a culture, expressing the polypeptide in the cultured host cell, and isolating the polypeptide from the culture; (2) a method of detecting exposure of a fish to nervous necrosis virus by providing a serum sample from a fish, contacting the serum sample to a substrate coated with a polypeptide of the invention, and determining whether antibodies in the serum sample specifically bind to the polypeptide on the substrate, where antibodies specifically binding to the polypeptide on the substrate indicates that the fish has been exposed to the nervous necrosis virus; and (3) a method of eliciting an antibody response to a nervous necrosis virus in an animal (e.g., a fish) by administering a polypeptide, nucleic acid, or cell of the invention to an animal in an amount sufficient to elicit an antibody response to the nervous necrosis virus.
An xe2x80x9cisolated nucleic acidxe2x80x9d is a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are nucleic acids present in mixtures of different (i) DNA molecules, (ii) transfected cells, or (iii) cell clones: e.g., as these occur in a DNA library such as a cDNA or genomic DNA library.
The term xe2x80x9csubstantially purexe2x80x9d as used herein in reference to a given polypeptide means that the polypeptide is substantially free from other biological macromolecules. The substantially pure polypeptide is at least 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
A xe2x80x9cconservative amino acid substitutionxe2x80x9d is one in which an amino acid residue is replaced with another residue having a chemically similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
The xe2x80x9cpercent identityxe2x80x9d of two amino acid sequences or of two nucleic acids is determined using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990), modified as in Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3. Where gaps exist between two sequences, Gapped BLAST is utilized as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. See http://www.ncbi.nlm.nih.gov.
Other features or advantages of the present invention will be apparent from the following detailed description, and also from the claims.